The Orthomyxoviridae are a family of RNA viruses which infect vertebrates. The family includes those viruses which cause influenza.
Influenza is a viral infection of the respiratory system characterized by fever, cough, and severe muscle aches. There are three genera of influenza virus, identified by antigenic differences in their nucleoprotein and matrix protein: Influenzavirus A, Influenzavirus B and Influenzavirus C.
Influenza A and B viruses each contain eight segments of single stranded RNA (ssRNA). The viruses comprise major external virion proteins, haemagglutinin (H) and neuraminidase (N), of which there are 16 H subtypes and 9 N subtypes which probably form all 144 possible permutations.
Influenza C virus contains seven segments of ssRNA, because the virus lacks a separate neuraminidase gene (see Lamb, R. and Krug, R. M. (1996) Chapter 45; Orthomyxoviridae: The viruses and their replication—Fields Virology, 3rd Edition, Raven Publishers, Philadelphia).
The major causative agent of human influenza is the type A virus. The virus genome consists of eight negative sense, single-stranded RNA segments. The RNA encodes 9 structural and 2 non-structural proteins. These are known to encode the influenza virus proteins as set out below:                Segment 1 encodes the polymerase protein PB2        Segment 2 encodes the polymerase protein PB1        Segment 3 encodes the polymerase protein PA        Segment 4 encodes the haemagglutinin protein (HA).        Segment 5 encodes the neuraminidase protein (NA).        Segment 6 encodes the nucleoprotein (NP).        Segment 7 encodes two matrix proteins (M1 and M2).        Segment 8 encodes two non-structural proteins (NS1 and NS2).        
Human influenza viruses A and B are both responsible for seasonal disease in people, but only influenza A viruses cause worldwide pandemics. In human viruses, three distinct haemagglutinins, referred to as H1, H2, and H3 and two distinct neuraminidases, referred to as N1 and N2 have been identified. Viruses are classified by their constituent haemagglutinin and neuraminidase proteins into subtypes. For example, the viral strain which caused the “Spanish” flu pandemic of 1918 belongs to the H1N1 subtype. The H2N2 subtype appeared in 1957 and replaced H1N1; the H3N2 subtype appeared in 1968 and replaced H2N2. Each replacement event is known as an antigenic shift, and results in a pandemic as the entire human population lacks effective immunity to the new virus. Following a shift the major viral H and N surface proteins undergo continuous and progressive antigenic changes called antigenic drift. Drift viruses cause annual epidemics of influenza. Currently the drift descendents of H3N2 and H1N1 (which reappeared in 1977) are co-circulating. Influenza B virus does not cause pandemic influenza but contributes to epidemics.
However, the majority of influenza A viruses exist in various waterfowl, causing subclinical gut infections. For example, in October 2003, an epidemic of influenza in chickens began sweeping through several countries in the Pacific Rim (Vietnam, Thailand, Japan, China, South Korea, Cambodia), and has recently reached Europe. This virus is designated H5N1. The H5 molecule is common among bird influenza viruses but has not been found in influenza viruses that cause human epidemics. However, sporadic human cases of H5N1 (with an alarmingly-high fatality rate) have been occurring ever since and are of significant concern.
Genomic studies suggest that the human pandemic viruses arose from avian viruses adapting to humans (1918), or genetically interacting with an existing human virus (1957 and 1968). Thus, as avian viruses (such as H5N1 and H7N7) move from their natural host into domestic poultry and into close contact with humans, there is concern about an emerging new pandemic virus. However, none of these viruses currently transmits effectively from person-to-person. Highly infectious new pandemic viruses all cause high morbidity and mortality, with 50 million estimated worldwide deaths for 1918 virus and 1-5 million for 1957 and 1968 viruses. Although an influenza infection elicits a strong immune response against the strain that caused it, the speed with which new strains arise by antigenic drift soon leaves a previously infected individual susceptible to a new infection. Influenza vaccines have been available commercially for many years and include killed and live vaccines. Some vaccines contain inactivated virus particles or more usually just the purified H and N components. These vaccines have proved helpful in reducing the extent and severity of influenza epidemics. However, because of the phenomenon of antigenic drift, the influenza strains used as the basis of existing vaccines are reassessed from year to year by WHO and may have to be changed. Also, any new vaccine required for a new pandemic virus would take several months before it could be made available for administration.
Other lines of defense against influenza include antiviral drugs. For example, Amantadine and Rimantadine inhibit the action of one of the matrix proteins needed to get viral RNA into the cytosol. These drugs are effective against all influenza type A viruses (but not type B viruses) but a rapid evolution of resistance to the drugs has been observed.
Alternatively, Zanamivir (Relenza®) and Oseltamivir (Tamiflu®) block neuraminidase and thus act to inhibit the release of progeny virions from infected cells and the spread of infection. However, the effectiveness of these therapies is somewhat limited. Treatment has to be started soon after infection, it is given twice daily, and is only able to shorten the duration of symptoms by one to three days. Virus that is resistant to Tamiflu is being found in patients with influenza.
Another influenza pandemic is inevitable, and is expected to result in widespread morbidity and upwards of a million deaths worldwide, despite developments in vaccinology and antiviral drugs. New measures to combat influenza are urgently needed.
DI viruses have a long history. They were discovered as auto-interfering elements in influenza A virus preparations by von Magnus who studied them in the late 1940s and early 1950s (e.g. von Magnus, P (1947) Ark. Kemi. Mineral. Geol, 24b: 1). For many years these interfering elements were named after him. Later, when it was realized that these elements were found almost universally amongst viruses, they were called DI viruses (see e.g. Huang & Baltimore (1970) Nature 226: 325-327). Interest in DI viruses reached a peak in the 1970's but then waned due to an over-extravagant expectation of their in vivo antiviral activity.
All influenza A viruses appear to have a replication apparatus that allows the exchange of genome segments (reassortment) in dually infected cells, giving these viruses immense genetic flexibility. Such an event gave rise to the 1957 and 1968 pandemic viruses. In addition to the normal replication process, mistakes in replication occur that give rise to small RNAs of 400-500 nt lacking around 80% of the central sequence of the template, which appears to result from the polymerase copying the initial part of the template, detaching from the template and then rejoining and copying the other terminus. These small RNAs retain the terminal replication and encapsidation signals, and their small size suggests that more copies can be made in unit time compared with the full-length RNA segment. Encapsidation of genomic RNAs appears to be an organized process so that a virion contains just one copy of each of the 8 segments. A virion does not appear to discriminate between a defective and a full-length RNA, so when defective RNAs are in excess they are preferentially encapsidated. A particle containing the deleted genome segment cannot synthesize the viral protein(s) normally encoded by that RNA, and is non-infectious, although it can be replicated in trans when that cell is infected by an influenza A virus. Incorporation of defective RNAs into virions results in a reduction in the amount of infectious virus produced. Thus virions carrying a deleted genome were know as interfering or defective-interfering (DI) viruses.
Viruses of Orthomyxoviridae family therefore give rise spontaneously to defective RNA segments as a result of an internal deletion (75-80% of the nucleotides) in one or more genomic segments. The DI virus genome is therefore a deleted form of the genome of the infectious virus which gave rise to it; and it has several unique properties which distinguishes it from other types of defective viral nucleic acid molecules (see Dimmock, N. J. (1996) “Antiviral activity of defective interfering influenza virus in vivo”—Viral and other infections of the human respiratory tract; S. Myint and D. Taylor-Robinson (Eds), Chapman & Hall).
Compared to an active, i.e. live or infectious virus, a DI virus is non-infectious and replicates only when its genome is present in a cell which has been infected by a virus with a complete genome (sometimes referred to as a “helper virus”). DI influenza virus is encapsidated into virus particles which are usually indistinguishable in size and protein composition from infectious virus particles.
After arising, de novo, a DI genome is rapidly amplified in concentration relative to that of the genome of the infectious virus, so that within a few infectious cycles (or passages) there is more DI virus in a population than infectious virus.
DI virus has the ability to interfere intracellularly with infectious virus so that it is specifically able to inhibit multiplication of infectious virus.
In vivo animal studies have shown that spontaneously produced DI influenza A virus (A/equine/Newmarket/7339/79 (H3N8)) can, in sufficient amount, protect mice against lethal influenza A challenge with both the homologous virus (EQV) or with heterologous subtypes A/WSN (H1N1) or A/PR/8/34 (H1N1). In these studies the DI virus preparation was UV-treated in order to inactivate any live helper virus present. A single administration appeared to provide prophylaxis for up to about 5 days. However, these DI virus preparations were heterogeneous and comprised a multiplicity of undefined defective RNA sequences from different genomic segments (see Noble and Dimmock (1994) J. Gen. Virol. 75: 3485-3491).
DI virus A/WSN (H1N1) grown in embryonated eggs protected mice against lethal challenge with A/WSN (H1N1). Comparison of egg-grown DI virus RNA species with DI virus RNA extracted from surviving mouse lungs showed that there were 5 putative RNAs responsible for mouse survival. Each of the five RNA species of the DI virus had an internal deletion (see Noble & Dimmock (1995) Virology 210: 9-19). The 3′ and 5′ ends of four of these RNA species appeared intact.
Duhaut & Dimmock (2000, Virology 275: 278-285) modified a defective segment 1 RNA of EQV by placing it under the control of a human RNA polymerase I promoter (POLI) in a plasmid. Each of the plasmids encoded an RNA of approx 400 nucleotides but, due to the exact position of the internal deletion, differing lengths of the 5′ and 3′ end sequences remained. Vero cells were transfected with each plasmid together with one of three different helper virus subtypes, including the parent (H3N8) or an H2N2 or H1N1 subtype. Serial passage was carried out in cell culture. At least 150 nucleotides at the 5′ end of the DI virus RNA were found to be necessary for reliable passage in vitro in each of the cell lines used together with the particular helper viruses used.
It has not been possible to experimentally elucidate the process by which non-cloned DI influenza A viruses reduce the yield of infectious virus, inhibit virus-induced cytopathology, and protect animals from clinical disease, as most populations of DI virus contain many different defective RNA sequences, derived from different genome segments and with a variety of central deletions. Thus the RNA content of such non-cloned populations of defective virus cannot be reproduced effectively, and it has not been possible to analyse the relationship between RNA sequence and antiviral activity.
Duhaut & Dimmock (2002, J. Gen. Virol. 83: 403-411) demonstrated that a DI virus RNA derived from a plasmid system appears to behave authentically in cell culture. One plasmid (POLI-317) gave rise to DI virus RNA that replicated stably in vitro in the presence of helper virus and strongly inhibited the production of the helper virus in that system.
Duhaut & Dimmock (2003, J. Virol. Methods 108: 75-82) described the preparation of a defined (i.e. cloned) DI influenza A virus generated entirely from plasmids which were used to transfect host cells in culture. The plasmids used encoded the DI RNA (H3N8 or H7N7) and infectious influenza virus (A/WSN, H1N1). DI virus generated in this way was passaged once in embryonated chicken's eggs and then administered to mice in the presence of helper virus (H1N1). The cloned DI virus propagated intact into mouse lung. The cloned DI virus (without infectious helper) was also tested for any protective effect in mice against a lethal (H1N1) challenge. Some very weak and short lived prophylactic effect was observed, but this only delayed the onset of clinical symptoms and death in the mice.
Noble et al. (2004, Vaccine 22: 3018-3025) reported an in vivo study in mice using a naturally occurring (i.e. heterogeneous and undefined) DI virus preparation (EQV H3N8). Administration of this DI virus preparation to mice was found to generate prophylaxis protection for a period, and at the same time converted an otherwise lethal infection into an avirulent and immunizing infection.
Dimmock & Marriott (2006, J. Gen. Virol. 87: 1259-1265) described an apparent anomaly in which a heterogeneous and undefined DI virus preparation solidly protects mice from lethal disease caused by A/PR/8/34 (H1N1) and A/WSN/40 (H1N1) viruses, but only marginally protects from disease caused by A/Japan/305/57 (A/Jap H2H2). A/Jap was found to require 300-fold more infectious units to cause clinical disease in mice than A/PR8. The proportions of DI virus and challenge virus were varied and tested. A conclusion reached was that the efficacy of the DI virus depends on the infectious dose of challenge virus rather than its disease-causing dose.
Mann et al. (2006, Vaccine 24, 4290-4296) tested heterogeneous and undefined DI A/EQV RNAs that had been rescued by (A/PR8) in ferrets. DI virus was administered in two doses followed by challenge with infectious A/Sydney 5/97 (H3N2). Though the infectious challenge was not lethal, the DI virus-treated ferrets showed only occasional and mild clinical symptoms, compared to the control animals which became severely ill.
US2006/0057116 A1 (Kawaoka and Neumann) describes plasmids and a method of transfecting and culturing cells to produce recombinant influenza A virus in vitro in the absence of any helper virus. Specifically, influenza A viruses can be prepared entirely from their cloned cDNAs in transfected cell lines. Mutations can be incorporated into any gene segment.
WO2006/051069 (Solvay Pharmaceuticals & Erasmus University) discloses conditionally defective influenza virus particles and a method of making them. From the starting point of transfected cells not being able to produce large quantities of defective influenza virus particles for use as vaccines, the specification teaches an alternative method. The method involves a cell transfected with 7 RNA segments of the influenza virus and an eighth segment in which a polymerase encoding sequence is deleted. The cell includes a second expression plasmid carrying the sequence of the deleted polymerase. On expression, the transfected cell yields “conditionally” defective virus particles which can only replicate in a cell line expressing the polymerase that is not present in the defective genome. The defective influenza virus particles can only replicate once in suitable, albeit not complemented, host animals or cells. The conditionally defective virus particles are intended for vaccine use or gene delivery purposes and so advantageously the virus particle preparations are unable to replicate in normal cells and contain no wild-type or helper virus.
Although a prototype system has been described (see Duhaut & Dimmock, 2003 supra) for preparing a cloned DI influenza A virus (which turned out to be only weakly protective on one occasion in mice), it does not offer a practical route for preparing the necessary amounts of cloned DI viruses needed for further laboratory investigations, let alone the amount of cloned DI virus that would be needed on a routine basis in order to carry out animal and human clinical trials or provide for prophylaxis and/or therapy in routine, epidemic or pandemic situations.
von Magnus, P. (1951a) Acta Pathol Microbiol Scand 28, 250-277; von Magnus, P. (1951b) Acta Pathol Microbiol Scand 28, 278-293; von Magnus, P. (1951c) Acta Pathol Microbiol Scand 29, 157-181; and von Magnus, P. (1954) Adv Virus Res 21, 59-79 each describe standard (i.e. infectious) A/PR8 (H1N1) virus made by inoculation of embryonated chickens eggs with “allantoic fluids diluted 10−6.” On page 158 of von Magnus (1951a) incomplete virus (i.e. DI virus) was made “by serial passages of undiluted allantoic fluids” with “1st, 2nd, 3rd, etc passages of undiluted virus.” Up to 4 passages were made.
Fazekas de St Groth, S. & Graham, D. M. (1954). “The production of incomplete influenza virus particles among influenza strains. Experiments in eggs.” Brit J Exp Path 35, 60-74. Also, von Magnus, P. (1965) “The in ovo production of incomplete virus by B/Lee and A/PR8 influenza viruses.” Arch Virol 17, 414-423. These references describe the production of incomplete (DI) B/Lee virus in embryonated chickens eggs. Production usually required 6 or more passages of undiluted virus.
Meier-Ewert, H. & Dimmock, N. J. (1970). “The role of the neuraminidase of the infecting virus in the generation of noninfectious (von Magnus) interfering virus.” Virol 42, 794-798. This reference describes the production of incomplete (DI) A/Jap/305/57 (H2N2) virus. Table 2 shows how the virus production required 3 serial undiluted passages.
Rott, R. & Schafer, W. (1960) “Untersunchungen uber die hamaggluttinierenden-nichtinfektiosen Teilchen der Influenza-Viren. I. Die Erzeugung von ‘inkompletten Formen’ beim Virus der klassischen Geflugelpest (v. Magnus Phanomen)” Zeitschrift fur Naturforschung 16b, 310-321; and Carter, M. J. & Mahy, B. W. J. (1982). Arch Virol 71, 12-25. These references describe how incomplete A/fowl plague virus (H7) was produced by serial passage of culture fluids at high multiplicity—usually undiluted virus. The cell culture fluids were obtained from chick embryo fibroblast cells.
Huang, A. S. & Baltimore, D. (1970) “Defective viral particles and viral disease processes” Nature (Lond) 226, 325-327. This review article at page 325 describes how the synthesis of DI particles by cells or animal tissues on infection with high multiplicities (or undiluted passage virus) is achieved for Rift Valley fever virus, vesicular stomatitis virus, fowl plague virus, simian virus 40, polyoma virus, lymphocytic choriomeningitis virus, Sendai virus, simian virus 5, and poliovirus.
Holland, J. J. (1990a) “Defective viral genomes” In Virology, 2nd edn, pp. 151-165. Edited by B. N. Fields & D. M. Knipe. New York: Raven Press. In this review article, page 155 describes how serial undiluted passage of virus in cell culture (or eggs or animals) is still the method of choice for generation of DI particles of any virus.
Holland, J. J. (1990b) “Generation and replication of defective viral genomes” In Virology, 2nd edn, pp. 77-99. Edited by B. N. Fields & D. M. Knipe. New York: Raven Press. Referring to FIG. 2 this book chapter discloses how DI particle bands did not appear until the fourth (undiluted virus) passage.
Nayak, D. P., Chambers, T. M. & Akkina, R. K. (1985) “Defective-interfering (DI) RNAs of influenza viruses: origin, structure, expression and interference” Curr Topics Microbiol Immunol 114, 103-151 is a review article which attests to the production of DI viruses by serial independent undiluted passage of virus.
The cloned DI influenza A virus produced in cell culture does not provide sufficient quantities of cloned virus for practical application. A problem that the invention seeks to solve is how to produce sufficient virus for in vivo studies and for pharmaceutical uses.
The inventor attempted to produce cloned DI influenza A virus by passage in embryonated hens' eggs, but too low a yield of DI virus resulted. A problem that the present invention seeks to solve is therefore how to provide sufficient yield of cloned DI influenza A virus by passage in embryonated eggs.